In most types of cancer, the formation of distant metastasis is the point at which the disease becomes lethal. At present, there is no clinical method to detect metastatic dissemination and colonization of distal sites until radiologically evident, at which point the function of the organ has already been compromised. The Shea lab has developed a biomaterial implant that recruits metastatic cancer cells in xenogeneic human and syngeneic mouse models of breast cancer. Scaffold implantation has facilitated detection of metastasis prior to colonization of organs and has been shown to reduce metastatic disease burden, ultimately resulting in enhanced survival with surgical intervention. This survival benefit associated with scaffold implantation in animal models is hugely promising, however the mechanism behind this benefit is not currently understood. Mechanistic understanding of the underpinnings of this observation will provide knowledge that will suggest if this observation can be expected in humans, and will thus inform the design of planned clinical trials. Furthermore, understanding of the mechanism underlying the observed survival benefit will also provide unprecedented evidence for how metastasis can be manipulated at a physiological systems level and inform other therapeutic targets and interventions. The specific aims of this proposal will test two hypotheses for the demonstrated therapeutic benefit. Aim 1 will quantify phenotypic heterogeneity of implant-captured tumor cells and investigate the hypothesis that these cells represent a highly plastic or stem-like population. This will be tested via isolation of scaffold captured tumor cells, circulating tumor cells (CTCs), primary tumor cells, and metastatic tumor cells in an organ (lung, liver or bone). The phenotype of cells from different locations will be compared via isolation of RNA and subsequent targeted q-RT-PCR to study differential expressions of genes associated with stemness, metastasis, differentiation, the poor-prognosis gene signature, and the epithelial to mesenchymal transition between different subpopulations. These differences will also be validated via functional assays in vitro such as migration, invasion, transendothelial migration, and mammosphere formation assays. Aim 2 will investigate the hypothesis that the scaffold immune microenvironment influences tumor cell phenotype including stemness and plasticity. This will be tested via immunomodulation of the scaffold microenvironment in vivo via lentiviral delivery of immunomodulatory factors and subsequent analysis of immune cell recruitment and subsequent tumor cell recruitment and phenotype. This data set will allow correlation of the immune composition of a pre-metastatic niche with the efficiency of tumor cell capture of that niche as well as how the niche influences tumor cell phenotype. Overall, biomaterial scaffolds capable of recruiting metastatic tumor cells in vivo represent a transformative approach that can not only to serve as a platform for early detection and intervention, but also serves as a defined site in vivo to study metastasis as a physiological systems level event.